Molecular Haplotyping by Real-time PCR
Genevieve Pont-Kingdon, Alison Millson and Elaine Lyon
from: Real-Time PCR: Current Technology and Applications (Edited by: Julie Logan, Kirstin Edwards and Nick Saunders). Caister Academic Press, U.K. (2009)
Abstract
Molecular real-time PCR methods can determine whether two or more mutations are on the same or different chromosomes. This ability to haplotype without family studies is useful for research and clinical purposes and can give an advantage over genotyping. Haplotyping by real-time PCR with hybridization probes has been demonstrated for adjacent repeats and single base alterations, with a probe that covers both sites. However, base alterations may be separated by distances greater than a traditional hybridisation probe will cover. We described a probe design that covers both (or all) sites, but does not include the entire sequence between the sites. When hybridized with the template, the template is forced to form a loop. This "loop-out" probe will dissociate from the template as a unit, therefore allowing haplotyping of base alterations separated by over 80 bp. Examples of haplotyping by traditional probes for adjacent sequence variants, as well as examples of "loop-out" probes are presented read more ...
